[Analysis of gene regulation in vertebrates has long benefited from studies initiated with the p-globin locus. Fundamental principles of developmental stage- and tissue-specific control of transcription originated with the study of this complexly regulated locus. The overall goal of my research has been to understand basic regulatory mechanisms governing human p-like globin gene switching, in this instance, autonomous yglobin gene silencing during the adult stage of definitive erythropoiesis. We recently identified a novel Ayglobin gene silencer motif and an associated represser complex that are linked to the first new HPFH point mutation to be described in over 10 years. This silencer is located at -570 relative to the mRNA CAP site in a GATA binding motif and repression is mediated by GATA-1 binding at this site. Mutation of this site in our (3-YAC transgenic mouse model resulted in a HPFH phenotype. This phenotype is corroborated by the discovery of HPFH in a family with the analogous mutation in the Gy-9'c-bin gene (Luo et al., 2004). The proposed study will test the hypothesis that y-globin gene silencing is mediated, in part, through GATA-1 recruitment of co-repressors. We will assess the function of the -570 silencer in vivo, characterize the GATA-1-Mi2 represser complex that binds at this site and identify its component proteins. Several HPFH mutations are associated with a loss of GATA-1 binding. We will test the universality of this mechanism in vivo. Finally, we will generate and identify novel represser binding sites within the Ay-globin gene promoter using a cell-based reporter assay to select for HPFH mutations. These aims will extend our studies identifying the role of various c/s-regulatory motifs from the previous funding period into functional studies that will discern molecular mechanisms of y-globin gene silencing by trans-acting factors comprising represser complexes. These proteins may be developed into potential therapeutic targets to increase HbF.] Sickle cell disease (SCO), p-thalassemias and Cooley's anemia are common genetic diseases that affect millions of people worldwide. SCO alone impacts one of 500 African Americans born each year. Understanding the molecular mechanisms controlling globin gene switching may aid in the development of targeted therapies or therapeutics to treat these diseases, particularly research aimed at turning on the fetal y-globin genes, which has been shown to be effective for the treatment of SCO.